Expression of heterologous proteins in yeast after transformation of yeast cells with suitable expression vectors comprising DNA sequences coding for said proteins has been successful for many species of polypeptides, such as glucagon, glucagon like peptides and their functional analogues. Yeasts, and especially Saccharomyces cerevisiae, are preferred host microorganisms for the production of pharmaceutically valuable polypeptides due to the stable yield and safety.
However, it is often found that the expression product is a heterogeneous mixture of species of the desired polypeptide precursor having different amino acid chain lengths. A number of proteases, activated by the PEP4 gene product are responible for yeast protein degradation. Mutation in the PEP4 gene such as the pep4-3 mutation is often used to reduce cellular proteolysis whereby the quality and yields of heterologous proteins of interest can be improved. EP 341215 describes the use of a yeast strain that lacks carboxypeptidase ysca activity for the expression of the heterologous protein hirudin. Wild-type yeast strains produce a mixture of desulphatohirudin species differing in the C-terminal sequence due to the post-translational action of endogeneous yeast proteases on the primary expression product. It is shown that yeast mutant strains lacking carboxypeptidase ysc.alpha. activity are unable to remove amino acids from the C-terminus of heterologous proteins and therefore give rise to integral proteins.
The use of yeast strains defective in protease A, B, Y, and/or S activity can only partially reduce random proteolysis of foreign gene products.
Another problem encountered in production of heterologous proteins in yeast is low yield, presumably due to proteolytic processing both in intracellular compartments and at the plasma membrane caused by aberrant processing at internal sites in the protein e.g. secretion of human parathyroid hormone (Gabrielsen et al. Gene 90: 255-262, 1990; Rokkones et al. J. Biotechnol. 33: 293-306, 1994), and secretion of .beta.-endorphine by S. cerevisiae (Bitter et al. Proc. Natl. Acad. Sci. USA 81: 5330-5334, 1984). Some polypeptides, e.g. polypeptides having from about 10 to about 55 amino acids or shorter chains and none or a few disulphide bonds and/or are rich in basic amino acids, such as .beta.-endorphine, glucagon and glucagon like peptides may be especially susceptible to intracellular and extracellular proteolytic degradation when expressed in a heterologous host due to their short-chain open and non-stable structure resulting in an inhomogeneous product.
WO 95/23857 discloses production of recombinant human albumin (rHA), which is a very large carrier-type protein cross-linked with 17 disulphide bonds and having a molecular weight of about 66 kD, in yeast cells having a reduced level of yeast aspartyl protease 3 (Yap3p) proteolytic activity resulting in a reduction of undesired 45 kD rHA fragment and in a 30 to 50% increased yield of recovered rHA produced by the haploid .DELTA.yap3 yeast strain compared to the rHA produced by the corresponding haploid YAP3 wild-type yeast strain.
Previously, Bourbonnais et al. (Biochimie 76: 226-233, 1994), have shown that the YAP3 protease gene product has in vitro substrate specificity which is distinct though overlapping with the Kex2p substrate specificity, and shown that Yap3p cleaves exclusively C-terminal to arginine residues present in the prosomatostatin's putative processing sites. Moreover, Cawley et al. (J. Biol. Chem. 271: 4168-4176, 1996) have determined the in vitro specificity and relative efficiency of cleavage of mono- and paired-basic residue processing sites by Yap3p for a number of prohormone substrates, such as bovine proinsulin.
The purpose of the present invention is to provide an improved method for the production of secreted polypeptides having up to about 55 amino acids, preferably from 10-50 amino acids, more preferably from 15-40 or preferably from 25-35 amino acids in the polypeptide chain, and having from 0 to 3 disulphide bonds, preferably no more than one disulphide bond, in the structure in-a yeast expression system. Preferred examples of polypeptides are glucagon and glucagon like peptides, CRF, and truncated and/or C-or N-terminally truncated and/or N-terminally extended forms of cocaine amphetamine regulated transcript (CART). Preferably, the production of polypeptides according to the invention is considerably increased, e. g. more than two fold compared to the production of said polypeptides in conventional yeast expression systems.
Often it is advantageous to produce heterologous polypeptides in a diploid yeast culture, because possible genetical defects may become phenotypically expressed in a haploid yeast culture, e.g. during continuous fermentation in production scale, and because the yield may be higher (Fu et al. Biotechnol. Prog., 12: 145-148, 1996; Mead et al. Biotechnol. Letters, 8: 391-396, 1986).
It would be obvious for a person skilled in the art to use the method of the present invention to produce other polypeptides satisfying the above criteria, such as insulin and insulin analogues, adrenocorticotropic hormones, angiotensinogen, atrial natriuretic peptides, dynorphin, endorphines, galanin, gastrin, gastrin releasing peptides, neuropeptide Y fragments, pancreastatin, pancreatic polypeptides, secretin, vasoactiv intestinal peptide, growth hormone releasing factor, melanocyte stimulating hormone, neurotensin, adrenal peptide, parathyroid hormone and related peptides, somatostatin and related peptides, brain natriuretic peptide, calcitonin, corticotropin releasing factor (CRF) (SEQ ID NO: 3), thymosin, and urotensin; and homologous or otherwise related peptides and fragments of these and other polypeptides (e.g. EEID-CART.sub.55-102 (SEQ ID NO: 2)), as long as the criteria of having up to 55 amino acids, preferably from 10-50 amino acids, more preferably from 15-40 or from 25-35 amino acids in the polypeptide chain, and having from 0 to 3 disulphide bonds, preferably no more than one disulphide bond in the structure, is fulfilled.